title: Ectopic expression of a truncated CD40L protein from synthetic post-transcriptionally capped RNA in dendritic cells induces high levels of IL-12 secretion.
abstract: BACKGROUND
  RNA transfection into dendritic cells (DCs) is widely used to achieve antigen expression as well as to modify DC properties. CD40L is expressed by activated T cells and interacts with CD40 receptors expressed on the surface of the DCs leading to Th1 polarization. Previous studies demonstrated that ectopic CD40L expression via DNA transfection into DCs can activate the CD40 receptor signal transduction cascade. In contrast to previous reports, this study demonstrates that the same effect can be achieved when RNA encoding CD40L is electroporated into DCs as evidenced by secretion of IL-12. To achieve higher levels of IL-12 secretion, a systematic approach involving modification of coding and noncoding regions was implemented to optimize protein expression in the DCs for the purpose of increasing IL-12 secretion.
  
  
  RESULTS
  Site-directed mutagenesis of each of the first five in-frame methionine codons in the CD40L coding sequence demonstrated that DCs expressing a truncated CD40L protein initiated from the second methionine codon secreted the highest levels of IL-12. In addition, a post-transcriptional method of capping was utilized for final modification of the CD40L RNA. This method enzymatically creates a type I cap structure identical to that found in most eukaryotic mRNAs, in contrast to the type 0 cap incorporated using the conventional co-transcriptional capping reaction.
  
  
  CONCLUSIONS
  The combination of knocking out the first initiation methionine and post-transcriptional capping of the CD40L RNA allowed for approximately a one log increase in IL-12 levels by the transfected DCs. We believe this is a first report describing improved protein expression of post-transcriptionally capped RNA in DCs. The post-transcriptional capping which allows generation of a type I cap may have broad utility for optimization of protein expression from RNA in DCs and other cell types.
authors.affiliation: Research and Development Department, Argos Therapeutics Inc, Durham, NC, USA. itcherepanova@argostherapeutics.com
authors.forename: Irina Y
authors.initials: IY
authors.lastname: Tcherepanova
authors.forename: Melissa D
authors.initials: MD
authors.lastname: Adams
authors.forename: Xiaorong
authors.initials: X
authors.lastname: Feng
authors.forename: Atsushi
authors.initials: A
authors.lastname: Hinohara
authors.forename: Joe
authors.initials: J
authors.lastname: Horvatinovich
authors.forename: David
authors.initials: D
authors.lastname: Calderhead
authors.forename: Don
authors.initials: D
authors.lastname: Healey
authors.forename: Charles A
authors.initials: CA
authors.lastname: Nicolette
mesh_terms: D020121:5' Untranslated Regions / Q000737:chemistry; D000595:Amino Acid Sequence; D001483:Base Sequence; D019013:CD40 Antigens / Q000502:physiology; D023201:CD40 Ligand / Q000096:biosynthesis / Q000235:genetics*; D002478:Cells, Cultured; D018387:Codon, Initiator / Q000235:genetics; D003713:Dendritic Cells / Q000378:metabolism*; D018664:Interleukin-12 / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012315:RNA Caps / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D015854:Up-Regulation
chemical_list: D020121:5' Untranslated Regions; D019013:CD40 Antigens; D018387:Codon, Initiator; D012315:RNA Caps; D023201:CD40 Ligand; D018664:Interleukin-12